A Novel Anti-Microbial Peptide from Pseudomonas, REDLK Induced Growth Inhibition of Leishmania - The Korean Journal of Parasitology
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ISSN (Print) 0023-4001
ISSN (Online) 1738-0006
Korean J Parasitol Vol. 58, No. 2: 173-179, April 2020
▣ BRIEF COMMUNICATION https://doi.org/10.3347/kjp.2020.58.2.173
A Novel Anti-Microbial Peptide from Pseudomonas,
REDLK Induced Growth Inhibition of Leishmania
tarentolae Promastigote In Vitro
Yanhui Yu1,2,†, Panpan Zhao1,†, Lili Cao1,3, Pengtao Gong1, Shuxian Yuan3, Xinhua Yao3, Yanbing Guo3, Hang Dong3,
Weina Jiang4,*
1
Key Laboratory of Zoonosis Research by Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun 130062, China; 2Clinical
laboratory, the Second Hospital of Jilin University, Changchun 130000, China; 3Jilin Academy of Animal Husbandry and Veterinary Medicine,
Changchun 130062, China; 4Department of Pathology, Qingdao Municipal Hospital, Qingdao 266071, China
Abstract: Leishmaniasis is a prevalent cause of death and animal morbidity in underdeveloped countries of endemic
area. However, there is few vaccine and effective drugs. Antimicrobial peptides are involved in the innate immune re-
sponse in many organisms and are being developed as novel drugs against parasitic infections. In the present study, we
synthesized a 5-amino acid peptide REDLK, which mutated the C-terminus of Pseudomonas exotoxin, to identify its ef-
fect on the Leishmania tarentolae. Promastigotes were incubated with different concentration of REDLK peptide, and the
viability of parasite was assessed using MTT and Trypan blue dye. Morphologic damage of Leishmania was analyzed by
light and electron microscopy. Cellular apoptosis was observed using the annexin V-FITC/PI apoptosis detection kit, mi-
tochondrial membrane potential assay kit and flow cytometry. Our results showed that Leishmania tarentolae was sus-
ceptible to REDLK in a dose-dependent manner, disrupt the surface membrane integrity and caused parasite apoptosis.
In our study, we demonstrated the leishmanicidal activity of an antimicrobial peptide REDLK from Pseudomonas aerugi-
nosa against Leishmania tarentolae in vitro and present a foundation for further research of anti-leishmanial drugs.
Key words: Leishmania tarentolae, Pseudomonas aeruginosa, leishmaniasis, antimicrobial peptide, REDLK
Leishmaniasis is a vector-borne disease caused by protozoan vaccine to treat with leishmaniasis.
parasites, genus Leishmania [1]. There are over 2 million new Antimicrobial peptides (AMPs) are generally considered as
cases of leishmaniasis per year, with approximately 400 mil- positively charged small peptides, comprising about 12-50
lion people at risk worldwide. It is also one of the most preva- amino acids. They have hydrophobic and hydrophilic regions,
lent causes of death and animal morbidity in endemic area of which make them soluble in aqueous environments, facilitat-
underdeveloped countries [2,3]. Leishmaniasis has different ing their interaction with biological membranes [8,9]. The
clinical manifestations, including cutaneous, mucocutaneous, therapeutic potential of AMPs, produced by a wide variety of
diffuse cutaneous, viscera and post kala-azar dermal leishman- organisms ranging from bacteria to animals, is enhanced ow-
iasis [4,5]. No effective vaccine against leishmaniasis is avail- ing to the ability of these compounds to rapidly kill a large
able and treatments drugs including pentavalent antimonials, number of micro-organisms [10]. Previous studies were detect-
amphoteric B, miltefosine and paromomycin, priced, have ed various types of AMPs with different structures, including
toxic side effects. Moreover, drug resistance to current treat- defensin, cecropins, magainins, and cathelicidins. Many AMPs
ments has been emerging [6,7]. Therefore, there is an urgent present broad activities against pathogens, including bacteria,
need for developing a safe, effective, and affordable drugs or viruses, fungi, and parasites [11]. The confirmed mechanisms
of AMPs include the disruption of microbial cellular mem-
• Received 20 November 2019, revised 20 March 2020, accepted 22 March 2020.
* Corresponding author (602096848@qq.com) branes, modulation of the innate and adaptive immunity [12].
†
These authors contributed equally to this paper. Pseudomonas exotoxin (PE) is a protein toxin that consists
© 2020, Korean Society for Parasitology and Tropical Medicine
of 3 functional domains, including the receptor binding do-
This is an Open Access article distributed under the terms of the Creative Commons
Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0) main I, the translocation domain II, and the enzymatic do-
which permits unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited. main III. The receptor binding domain can be genetically
173
174 Korean J Parasitol Vol. 58, No. 2: 173-179, April 2020 modified to create an immunotoxin by replacing the binding The viability of REDLK-treated Leishmania tarentolae was as- domain with antibodies specific for various cancers, leading to sessed using 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo- the killing of target cells by inhibition of protein synthesis via lium bromide (MTT; Sigma–Aldrich, St. Louis, Missouri, USA), ADP-ribosylation of elongation factor 2 and induction of as previously described [18]. Briefly, promastigotes were seeded apoptosis. Additionally, PE has certain toxicity to prokaryotic at 3 × 106 cells per well into a 96-well flat-bottom microtiter cells due to the slow growth of Escherichia coli expressing the plate and exposed to 12.5 µg/ml, 25 µg/ml, and 50 µg/ml recombinant immunotoxin [13,14]. Mutation analysis of the REDLK at 26˚C for 72 hr. PBS was used as the control. The plate C-terminus of PE has demonstrated that the last amino acids was centrifuged at 3,500 rpm for 15 min, re-suspended in 100 (positions 609-613) in REDLK are crucial to its cytotoxic activ- µl fresh medium, and treated with 10 µL of 5 mg/ml MTT at ity but unrelated to the ADP-ribosylation activity [15]. There- 26˚C for 4 hr. Each group was repeated 3 times. Absorbance fore, we were prompted to synthesize the short peptide was read at 570 nm to determine the amount of formazan pro- REDLK, a 5-amino acid peptide composed of arginine, glu- duction, which correlates with relative cell viability. Values were tamic acid, aspartic acid, leucine and lysine, to evaluate its bio- normalized by the untreated control. Cell viability (%)= A570 nm activities as an AMP. of treated cells/ A570 nm of control× 100. In this study, we used non-pathogenic Leishmania tarentolae Parasites were seeded into a 96-well flat-bottom microtiter (L. tarentolae), a parasite isolated from the gecko Tarentolae an- plate at 5× 106/well in the presence of 12.5 µg/ml, 25 µg/ml, nularis, as an in vitro model for evaluation of the specific leish- 50 µg/ml REDLK or PBS as a control. Reactions were per- manicidal activity of REDLK. Anti-microbial peptide, REDLK formed in triplicate wells per concentration. After 72 hr, viable derived from Pseudomonas aeruginosa is a promising candidate parasite counts were determined using a hemocytometer fol- as a novel antimicrobial peptide REDLK against Leishmania lowing trypan blue dye. tarentolae and lay a foundation for further research on studying L. tarentolae promastigotes were treated with different con- anti-leishmanial drugs. centrations (12.5 µg/ml, 25 µg/ml, and 50 µg/ml) of REDLK The UC strain of Leishmania tarentolae (ATCC PRA-229, or PBS at 26˚C for 72 hr. For light microscopy, 10 µl of each USA) promastigote conserved by our laboratory was cultured sample was loaded onto a glass slide, fixed with methanol, in BHI medium supplemented with 1% penicillin-streptomy- and stained with Giemsa for 2 hr. For electron microscopy, the cin and 5 µg/ml hemin (Sigma, St. Louis, Missouri, USA) at ultrathin sections were prepared as previously described [19] 26˚C. GFP-transfected Leishmania tarentolae was constructed and observed using a transmission electron microscope (Hita- using Leishmania expression vector pLEXSY-neo2 (Jena Biosci- chi, Tokyo, Japan). ence, Thuringia, Germany), as previously described [16]. The After L. tarentolae promastigotes were treated with different GFP expression cassette was integrated into the chromosomal concentrations of REDLK, the apoptotic cells were identified ssu locus of Leishmania tarentolae via homologous recombina- using the Annexin V-FITC/PI Apoptosis Detection Kit (KeyGen, tion. GFP can be stably expressed in promastigote. Nanjing, China) and a mitochondrial membrane potential as- REDLK is a 5-amino acid peptide (REDLK, mass: 659.36 say kit (JC-1) (Beyotime, Jiangsu, China), according to the Da). It was synthesized using standard Fmoc solid phase pep- manufacturer’s protocols [20]. JC-1, a lipophilic dye, which tide synthesis strategy on a Symphony XTM Multiplex Synthe- forms aggregates in mitochondrial matrix and emits red fluo- sizer (Rainin, Woburn, Massachusetts, USA). Fmoc was used rescence at 585 nm in normal cells. However, when mitochon- for the Nα protecting group and acid-labile tert-butyl-based drial membrane potential collapses, JC-1 de-aggregates as groups were used for side chain protection. Both auxiliary nu- monomers and emits green fluorescence at 514 nm. Carbonyl cleophile and in situ coupling reagent were used to facilitate cyanide m-chlorophenyl hydrazone (CCCP), which can cause the peptide bond formation [17]. Then, REDLK was purified quick mitochondrial membrane depolarization, was used as by reversed phase high performance liquid chromatography the positive control [21]. L. tarentolae promastigotes were cul- (RP-HPLC) and confirmed by mass spectroscopy (Supplemen- tured in BHI media with 12.5 µg/ml, 25 µg/ml, and 50 µg/ml tary Fig. S1) and amino acid analysis. Purified peptides were REDLK or PBS for 72 hr. The cells were stained with JC-1 dye. lyophilized and re-suspended in sterile PBS at a concentration Flow cytometry analysis was performed in a FACSCalibur flow of 1 mg/ml and stored at 4˚C until use. cytometer (BD Bioscience, San Jose, California, USA), and the
Yu et al.: Leishmanicidal activity of Pseudomonas peptide REDLK 175
data were analyzed using BD CellQuest Pro software. High levels of amorphous staining were likely due to release
Differences between the experimental group and control by cellular material (Fig. 2Ac, d).
group were determined using a Student’s t test. A one-way Transmission electron microscopy of AMP-treated parasites
analysis of variance (ANOVA) was used for multiple group revealed cytoplasm blebbing, a severe disruption of the mem-
comparisons. P-values of less than 0.05 were considered statis- brane structure and near complete loss of electron dense cyto-
tically significant. plasmatic components (Fig. 2B).
The cell viability was determined by Trypan blue dye test
and measuring the reduction of substrate MTT. REDLK inhib- 120
ited the growth of Leishmania tarentolae promastigotes by 3.2%,
100
39.5%, and 90.7% at the concentrations of 12.5 µg/ml, 25 µg/
Parasites survival (%)
ml, and 50 µg/ml, respectively after 72 hr treatment (Fig. 1). 80
MTT viability assay also produced similar results (Supplemen- 60
tary Fig. S2). *
40
Microscopic observation showed that REDLK can cause dra-
20
matic changes in the structural integrity of the parasite. Un- **
treated promastigotes presented thin, elongated cells with a 0
Control 12.5 25 50
unipolar flagellum and a clearly visible nucleus and kineto- REDLK (μg/ml)
plast (Fig. 2Aa, b). However, REDLK-treated promastigotes ex-
Fig. 1. REDLK-mediated killing effect on Leishmania tarentolae.
hibited rounding and swelling of cell bodies, with clear dis- Leishmania tarentolae promastigotes were incubated with differ-
ruption of the cellular membrane accompanied by release of ent concentrations of REDLK at 26°C for 72 hr. The promasti-
cytoplasmic contents into the medium. After 72 hr of expo- gotes were counted using a hemocytometer after trypan blue
dye. The promastigotes were susceptible to REDLK in a dose-
sure, nearly all of the treated promastigotes had lost their elon- dependent manner. Each column represents a mean of 4 repli-
gated form and appeared in various states of disintegration. cates. Error bar represents mean± SE . *P < 0.05, **P < 0.01.
A a b B a b
c d c d
Fig. 2. Morphologic changes of Leishmania tarentolae promastigotes induced with REDLK. (A) Optical microsgraphs of REDLK-treated
Leishmania tarentolae promastigotes. Untreated (a, b) or REDLK-treated (c, d) Leishmania tarentolae promastigotes were Giemsa-
stained after 72 hr exposure to 50 µg/ml peptide REDLK. Scale bar= 10 μm. (B) Transmission electron micrographs of REDLK-treated
Leishmania tarentolae promastigotes. Parasites were cultured in media with PBS (a), 12.5 µg/ml (b), 25 µg/ml (c), or 50 µg/ml (d) REDLK
for 72 hr. Membrane disruption, cytoplasm blebbing, and membrane breakages and depletion of electron-dense cytoplasmic material
are observed. Scale bar= 1 µm.
176 Korean J Parasitol Vol. 58, No. 2: 173-179, April 2020
A 104 b 10
4
a
103 103
102 102
101 101
100 100
100 101 102 103 104 100 101 102 103 104
d 10
4
c 104
103 103
102 102
101 101
100 100
100 101 102 103 104 100 101 102 103 104
B a 104 b 104 c 104
103 103 103
102 102 102
101 101 101
100 100 100
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
d 104 e 104 f 104
103 103 103
102 102 102
101 101 101
100 100 100
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
Fig. 3. Cellular apoptosis of REDLK-treated Leishmania tarentolae promastigotes. (A) Cellular membrane permeability and phosphatidyl-
serine exposure induced by REDLK in Leishmania tarentolae promastigotes. Parasites were cultured in media with PBS (a), 12.5 µg/ml
(b), 25 µg/ml (c), or 50 µg/ml (d) REDLK for 72 hr. The cells were stained with Annexin V-FITC and PI and analyzed by flow cytometer.
The results are representative of at least 3 independent experiments. (B) Depolarization of the mitochondrial membrane induced by
REDLK. (a) Untreated promastigotes not stained with JC-1. (b) Untreated promastigotes stained with JC-1. (c) CCCP-treated promasti-
gotes stained with JC-1. (d, e, and f) Promastigotes were treated with 12.5 µg/ml, 25 µg/ml, and 50 µg/ml REDLK and stained with An-
nexin V-FITC and PI. The results are representative of at least 3 independent experiments.
We co-stained REDLK-treated promastigotes with Annexin crosis or late-stage apoptosis.
V-FITC, which is a Ca2+-dependent phospholipid binding pro- On flow cytometer, the number of parasites staining posi-
tein with an affinity for PS, and propidium iodide (PI). PI can tive for PI, but negative for Annexin V (upper left quadrant),
permeate the protozoa and bind to cellular DNA only if a loss was significantly increased following exposure to 25 µg/ml
of plasma membrane integrity occurs at the same time as ne- and 50 µg/ml REDLK, compared to 12.5 µg/ml REDLK (Fig.Yu et al.: Leishmanicidal activity of Pseudomonas peptide REDLK 177 3A), showing that the peptide induced late-stage apoptosis or The REDLK peptide could inhibit the growth of promastigote necrosis of the parasite is dose-dependent. The peptide also by 3.2%, 39.5%, and 90.7% at the concentrations of 12.5 µg/ induced early-stage apoptosis (lower right quadrant). Howev- ml, 25 µg/ml, and 50 µg/ml, respectively. In previous report, er, when exposure to REDLK was increased to 50 µg/ml, the we observed leishmanicidal effect of KDEL that extracted from number of parasites during early-stage apoptosis was de- Pseudomonad exotoxin. The growth inhibition effect of REDLK creased as parasites transitioned from early-stage apoptosis to displayed significantly remarkable than that of KDEL peptide, late-stage apoptosis (Fig. 3A). These results demonstrate that which shares 10.4% and 33.3% growth inhibition at the con- REDLK has distinct effects on promastigotes in terms of mem- centrations of 25 µg/ml and 50 µg/ml, respectively [30]. brane permeability and PS accessibility. REDLK presented potent leishmanicidal activity against The loss of mitochondrial membrane potential is a key indi- Leishmania tarentolae in a dose-dependent manner. In all Leish- cator for the initiation of programmed cell death. After expo- mania species, the surface of promastigotes is surrounded by sure to different concentrations of REDLK, JC-1-stained para- glycosylphosphatidylinositol (GPI)-anchored proteins and sites led to a decrease in red fluorescence intensity (upper left metalloproteinase GP63. GP63 knockout mutants are more quadrant) and increase in green fluorescence intensity (lower susceptible to some types of AMPs and undergoes cellular right quadrant), compared to untreated parasites. These results apoptosis more readily than wild-type parasites [31,32]. GP63 suggest that REDLK can induce a loss of membrane electro- is inhibited in Leishmania tarentolae, which decreases the pre- chemical gradient in the mitochondria (Fig. 3B). vention ability of the peptide activity. Protozoan parasites of Leishmania parasites belong to the ge- There are at least 2 major mechanisms responsible for AMP- nus Leishmania and family Trypanosomatidae. They have a sin- mediated antimicrobial effects, including apoptotic and non- gle flagellum and a characteristic mitochondrial DNA-contain- apoptotic mechanisms [33]. In this study, the number of para- ing organelle. Infected female Phlebotomus sp., sandflies bites sites positive for PI, but negative for Annexin V, was signifi- vertebate host and could spread Leishmania parasites to verte- cantly increased following exposure to 25 µg/ml and 50 µg/ml brate hosts by bites. The promastigote flagellar form is found in REDLK, compared to 12.5 µg/ml, suggesting that late-stage the gut of the sandfly vector [22,23]. Leishmania tarentolae is a apoptosis of parasites induced by REDLK is dose-dependent. suitable model for the screening of potential anti-leishmanial REDLK can cause significant disruption of membrane integrity drugs in vitro, based on the following considerations: (i) More in Leishmania tarentolae promastigotes and ultimately result in than 90% of the Leishmania tarentolae genes are shared with the osmotic instability, vacuolar swelling, loss of cytosolic con- other pathogenic Leishmania species [24]; (ii) Leishmania taren- tents, and eventual cell death. REDLK-treated parasites pre- tolae do not induce clinical symptoms of leishmaniasis in sented positive for PI, but negative for Annexin V, which re- mammalian animals and is therefore safe for humans. Impor- sembled typical early apoptotic stage in which PS exposure oc- tantly, Leishmania tarentolae present similar sensitivity to pen- curred, but the membrane was not permeable. The early apop- tavalent antimony, amphotericin B, miltefosine and paromo- tosis in promastigotes was also confirmed by depolarization mycin as other Leishmania species that are pathogenic in hu- of mitochondrial membrane potentials. mans [6,25]. The availability of non-pathogenic species, L. In the present study, we demonstrated the leishmanicidal tarentolae provides us with a resource to do the preliminary in activity of a short peptide REDLK from Pseudomonas aeruginosa. vitro evaluation of novel anti-leishmanial drugs. REDLK induced apoptosis of L. tarentolate promastigote and AMPs are novel therapeutic agents that range from 12 to 50 resulted in parasite death. amino acids in length. As potent, broad spectrum antibiotics In summary, we evaluated the synthetic 5-amino acid pep- in all living organisms, AMPs permeabilized membranes and tide REDLK derived from from Pseudomonas aeruginosa as a modulation of the host immune responses [26]. In addition, novel anti-leishmanial agent against Leishmania tarentolae. The several amphibian AMPs, such as magainin, temporin, derma- action mechanisms of the peptide against Leishmania tarentolae septin, bombinin, and cecropin-melittin hybrid peptide, involve disruption of surface membrane integrity and cellular mammalian AMPs, including histatin-5, BMAP-28 and CM11, apoptosis. Our results provide the basis for further research on DRS 01 possess significant anti-leishmanial activity against in- studying anti-leishmanial drugs. tracellular amastigotes and extracellular promastigotes [27-29].
178 Korean J Parasitol Vol. 58, No. 2: 173-179, April 2020
ACKNOWLEDGMENTS 9. Ebenhan T, Gheysens O, Kruger HG, Zeevaart JR, Sathekge MM.
Antimicrobial peptides: their role as infection-selective tracers
for molecular imaging. Biomed Res Int 2014; 867381.
This work was, partly, supported by the National Natural
10. Lynn MA, Kindrachuk J, Marr AK, Jenssen H, Pante N, Elliott
Science Foundation of China (grant numbers 31672288, MR, Napper E, Hancock RR, Master WRM. Effect of BMAP-28
31302076, 31001057), National Science and Technology Major antimicrobial peptides on Leishmania major promastigote and
Project of the Ministry of Science and Technology of China amastigote growth: role of leishmanolysin in parasite survival.
(grant number 2012ZX09103301-021), China Postdoctoral Sci- PLoS Negl Trop Dis 2011; 5: e1141.
ence Foundation (No. 2014M552636), and the technological 11. Aguilar-Diaz H, Canizalez-Roman A, Nepomuceno-Mejia T,
Gallardo-Vera F, Hornelas-Orozco Y, Nazmi K, Bolscher JG, Car-
innovation and entrepreneurship projects of people returned
rero JC, Leon-Sicairos C, Leon-Sicairos N.Parasiticidal effect of
oversea in Jilin province of China (2012). synthetic bovine lactoferrin peptides on the enteric parasite
The authors thank Professor Xiaohuan Zou Institute of Mili- Giardia intestinalis. Biochem Cell Biol 2017; 95: 82-90.
tary Veterinary, Academy of Military Medical Sciences for pro- 12. Cederlund A, Gudmundsson GH, Agerberth B. Antimicrobial
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13. Müller F, Cunningham T, Beers R, Bera TK, Wayne AS, Pastan I.
CONFLICT OF INTEREST Domain II of Pseudomonas exotoxin is critical for efficacy of bo-
lus doses in a xenograft model of acute lymphoblastic leukemia.
No conflict of interest exists in this work, and the manu- Toxins 2018; 10: E210.
script was approved by all authors for publication. The work 14. Qaiser H, Aslam F, Iftikhar S, Farooq A. Construction and re-
described is original research has not been published previ- combinant expression of Pseudomonas aeruginosa truncated exo-
toxin A in Escherichia coli. Cell Mol Biol (Noisy-le-grand) 2018;
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64: 64-69.
15. Chaudhary VK, Jinno Y, Gallo MG, FitzGerald D, Pastan I. Mu-
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